2010 Circulation of Group 2 Coronaviruses in a Bat Species Common to Urban Areas in Western Europe

8 Pages·2010·0.36 MB·English

Circulation of Group 2 Coronaviruses in a Bat Species
Common to Urban Areas in Western Europe

Chantal B.E.M. Reusken,1 Peter H.C. Lina,2 Annemarie Pielaat,1 Ankje de Vries,1 Cecile Dam-Deisz,1

Jeroen Adema,1 Jan Felix Drexler,3 Christian Drosten,3 and Engbert A. Kooi4

Abstract

Fecal samples of 211 bats representing 13 different bat species from 31 locations in the Netherlands were analyzed
for the presence of coronaviruses (CoV) using a genus-wide reverse transcription (RT)–polymerase chain reaction.
CoVs are known for their high potential for interspecies transmission, including zoonotic transmission with bats as
reservoir hosts. For the first time, a group 2 CoV was found in a bat, Pipistrellus pipistrellus, in Europe. This is of
particular interest for public health as the reservoir host is a species that is common to urban areas in most of
Europe and notorious for its close interactions with humans. Four verspertilionid bat species were found to excrete
group 1 CoVs, viz. Myotis daubentonii, M. dasycneme, P. pipistrellus, and Nyctalus noctula. The last species is a newly
identified reservoir. The overall prevalence was 16.9% and positive bats were found at multiple widespread
locations. The circulating group 1 CoV lineages were rather species associated than location associated.

Key Words: Bats—Coronavirus— Field study—Public health—Zoonosis.

Introduction
T
he majority of emerging infectious diseases in
humans are zoonotic and emerging from wildlife reser-
voir hosts. Frequently, these zoonoses are caused by RNA
viruses (Jones et al. 2008). Recent examples are the influenza
A (H1N1) pandemic in 2009 (Dawood et al. 2009) and the
outbreak of severe acute respiratory syndrome (SARS) in
2002–2003, caused by the novel coronavirus SARS-CoV
(Drosten et al. 2003). The genus coronavirus (CoV) belongs to
the family of the Coronaviridae in the order of the Nidovir-
ales. CoVs are enveloped, positive-sense RNA viruses with
large genomes (29–32 kb) packaged in particles with corona-
like morphology (Holmes 2001). CoVs comprise three genet-
ically and serologically distinct groups (Gonzalez et al. 2003).
Group 1 and 2 viruses are pathogenic for mammals. Group 1
includes human CoV-229E (hCoV-229E) and hCoV-NL63.
Group 2 includes hCoV-OC43 and hCoV-HKU1. Group 3
viruses are foremost pathogenic for poultry.
CoVs are considered as a group of viruses with a high
potential for interspecies transmission. They are found in a
wide variety of hosts, including wildlife, livestock, poultry,
pets, and humans. Due to their long genomes, high recom-
bination frequency and high mutation rate, CoVs have the

potential to adapt to new host species (Woo et al. 2009). Ex-
amples of interspecies transmission of CoVs are the emer-
gence of porcine epidemic diarrhea CoV in swine in Europe
most likely from bats (Pensaert and de Bouck 1978, Vijay-
krishna et al. 2007), hCoV-OC43, which is believed to be the
result of a zoonotic transmission event of bovine CoV (Vijgen
et al. 2005), and SARS-CoV, which was shown to be intro-
duced into the human population from civet cats, which acted
as intermediate hosts, whereas bats are considered as the
primary reservoir hosts (Lau et al. 2005). Very recently, evi-
dence was found for a 208–322-year-old common ancestor for
a group 1 CoV and hCoV-229E circulating in Hipposideros bats
in Ghana (Pfefferle et al. 2009).
A large number of viruses have been detected in bats.
However, zoonotic transmission has only been shown for
rabies virus, bat lyssa viruses, Melaka reovirus, Ebola virus,
Marburg virus, Nipah and Hendra viruses, and SARS-like
CoV (Calisher et al. 2006, Wong et al. 2007). Bats (order
Chiroptera) constitute around 20% of all mammalian species
and are geographically widely dispersed with a wide variety
of habitats (Jones et al. 2002). Bats have a number of charac-
teristics that increase the likelihood of intra- and interspecies
transmission of viruses. They have, in general, a long life span,
they fly daily in pursuit of food, and some species fly long

1National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
2National Museum of Natural History ‘‘Naturalis,’’ Leiden, The Netherlands.
3Institute of Virology, University of Bonn Medical Centre, Bonn, Germany.
4Central Veterinary Institute of Wageningen University and Research, Lelystad, The Netherlands.

VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 10, Number 8, 2010
ª Mary Ann Liebert, Inc.
DOI: 10.1089=vbz.2009.0173

785

distances during seasonal migrations. Further, they show
gregarious roosting behavior, with up to several millions of
individuals per population. Roost sites include locations in
proximity to humans like houses (cellars and cavity walls),
stables, bridges, mines, and wells; frequent contact with in-
termediate hosts like domestic cats enlarge the possibility of
transmission of virus from bats (Calisher et al. 2006). Recent
studies indicate that bats are the natural hosts for all mam-
malian CoVs and that the circulation of CoVs in other mam-
mals is the result of occasional introductions from bats

(Vijaykrishna et al. 2007, Woo et al. 2009). Genetic analyses
show that bat CoVs belong to groups 1 and 2. Within group 2
they cluster in the subgroups 2b, which includes all SARS and
SARS-like CoV from different hosts, and 2c and 2d, which
represent two independent bat CoV subgroups.
To assess the putative risks for public health of bats as
reservoirs for CoVs with potential for interspecies transmis-
sion, we investigated to what extent CoVs circulate in a va-
riety of bat species and populations in the Netherlands.
Isolated bat CoV sequences were sequenced and analyzed

FIG. 1.
Distribution of sampled locations (a gray dot) and coronavirus (CoV)-positive locations (a black dot). Sampling
locations are indicated with a unique number: 1, Tollebeek; 2, Lisse; 3, Stokkelaarsbrug; 4, Almere; 5, Noordwijk aan Zee; 6,
Noordwijkerhout; 7, Wassenaar; 8, Scheveningen; 9, Stompwijk; 10, Den Haag; 12, Rijswijk; 14, Leidschendam; 15,
Waversveen; 16,’s Gravenland; 17,’s Gravenland; 18, Papenveer; 19, Ter Aar; 20, Woerdense verlaat; 21, Reeuwijk; 22, Gouda;
24, Doetinchem–De Huet; 25, Doetinchem–Wijnbergen; 26, Veldhuizen; 27, Beekermark-Zeddam; 28, Zeddam; 29,
Doetinchem–Slangenburg; 30, Maastricht-west; 31, Meerssen; 32, Berg en Terblijt; 33, Geulhem; 34, Sibbe.

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REUSKEN ET AL.

phylogenetically. To our knowledge this is the first report of
group 2 CoV circulation in bats in Europe. We show that
group 1 CoVs are commonly present in a variety of bat species
at multiple locations in the Netherlands. This includes the
identification of a new reservoir species.

Materials and Methods

Sampling

Bats were caught in several regions of the Netherlands
(Fig. 1). With exception of a few grounded bats, bats were
caught with mist nets in their foraging habitats in forests or
over water bodies (Haarsma and van Alphen 2009), and in the
southernmost part of the country at swarming sites at the
entrances of subterranean limestone quarries. After catching,
each bat was kept in a disposable cotton bag for the collection
of fecal pellets, after which the species, sex, age, and repro-
duction status were determined. All bats were released at
their capture site. The feces were collected with disposable
tweezers and stored at �208C till processing. All procedures
were in compliance with Dutch laws on animal handling and
welfare and wildlife conservation (Flora and Fauna Act li-
cense FF=75A=2003=169=a=b; Animal Ethics Clearance 802-
18230-00=2006025).

RNA isolation from bat feces

Feces
were resuspended
in HBSS
HANKS medium
(Invitrogen, Breda, The Netherlands). In accordance to spec-
imens availability, 10%, 5%, or 1% of fecal suspensions were
used. The fecal suspensions were centrifuged at 3000 g for
20 min, and 100 mL supernatant was used for extraction of
RNA. RNA was extracted as previously described (Boom et al.
1990). To check for cross-contamination, a negative control
sample was included for every four samples.

Reverse-transcription polymerase chain
reaction and sequencing

A nested CoV genus-wide reverse transcription (RT)-
polymerase chain reaction (PCR) was performed as described

previously (de Souza Luna et al. 2007). To exclude false-
negative results due to the presence of RT-PCR inhibiting
factors, each sample was also analyzed in the presence of a
CoV spike RNA at an experimentally predetermined detect-
able concentration. Only the results of samples that were
positive for CoV in the spiked RT-PCR were taken into ac-
count in this study. PCR amplicons were sequenced using an
ABI PRISM BigDye Terminator Cycle sequencing Ready Re-
action kit (Applied Biosystems, Nieuwerkerk a=d, Ijssel, The
Netherlands). All sequences were confirmed by sequencing
both strands. Sequences were edited using Kodon vs 3.5
(Applied Maths, St–Martens–Latem, Belgium).

Phylogenetic analysis

Nucleotide sequences were analyzed using Bionumerics vs
5.1 (Applied Maths). Evolutionary trees of nucleotide se-
quences were drawn using the maximum parsimony method.
The confidence values of the internal lineages were calculated
by performance of 1000 bootstrap analysis. Sequences from
the Dutch bat-CoVs are submitted in GenBank under acces-
sion nos. GQ259960-GQ259977. Accession numbers used in
the analysis are as follows: AY427798 (Toro virus); EU420138
(Miniopterus magnater, China, 1A); EF203064 (Rhinolophus si-
nicus, China, HKU2); EU375860 (Myotis dasycneme, Germany,
D3.10); EU375855 (M. dasycneme, Germany, D3.4); EU375855
(Pipistrellus nathusii, Germany, D5.16); EU375870 (Pipistrellus
pygmaeus, Germany, D5.85); EU375874 (Myotis daubentonii,
Germany, D8.38); EU375872 (M. daubentonii, Germany,
D8.45); DQ249224 (Myotis ricketii, China, HKU6); NC_002645
(hCoV-229E); NC_005831 (hCoV-NL63); DQ648854 (Rhinolo-
phus spp., China, A970); EU834953 (Rhinolophus megaphyllus,
Australia, 100); DQ249226 (M. magnater, China, HKU7);
NC_010438 (Miniopterus pusillus, China, HKU8); AY597011
(hCoV-HKU1); NC_005147 (hCoV-OC43); AY641576 (Avian
infectious brochitis virus); EF065515 (Rousettus lechenaulti,
China, HKU9=3); EF065514 (R. lechenaulti, China, HKU9=2);
DQ648856
(Rhinolophus
ferrumequinum,
China,
273);
DQ022305 (R. sinicus, China, HKU3); DQ648857 (Rhinolophus
macrotis, China, 279); AY304488 (Civet SARS-CoV-SZ16);
DQ071615 (Rhinolophus pearsoni, China, Rp3); EF065505

Table 1. Overview of Bat Species Tested for Coronaviruses

Species
Total no.
(positive)

No. of male
adult=juvenile
(positive)

No. of female
adult=juvenile
(positive)

No. of female
lactating=gravid
(positive)
Locationsa

Eptesicus serotinus
1 (0)
0 (0)=1 (0)
14
Myotis bechsteinii
4 (0)
4 (0)=0 (0)
30
Myotis brandtii
2 (0)
1 (0)=0 (0)
1 (0)
26, 32
Myotis daubentonii
50 (8)
17 (4)=2 (0)
20 (2)=2 (0)
9 (2)
6, 9, 12, 15, 20, 27, 30, 32, 33, 34
Myotis dasycneme
105 (20)
13 (1)=21 (7)
48 (9)=18 (3)
5 (0)
1, 3, 4, 9, 15, 18, 19, 20, 21, 22
Myotis emarginatus
6 (0)
5 (0)=0 (0)
1 (0)
30, 31, 32, 34
Myotis myotis
1 (0)
1 (0)=0 (0)
31
Myotis mystacinus
3 (0)
3 (0)
26, 28, 29
Myotis nattereri
2 (0)
2 (0)
26, 31
Nyctalus noctula
14 (5)
5 (2)=0 (0)
8 (2)
1 (1)
6, 7, 8, 10
Pipistrellus nathusii
8 (0)
8 (0)=0 (0)
2, 5, 6, 7
Pipistrellus pipistrellus
8 (2)
1 (1)=1 (1)
3 (0)
3 (0)
6, 17, 24, 25
Plecotus auritus
7 (0)
3 (0)=0 (0)
3 (0)
1 (0)
16, 17, 26, 27, 29, 31

aNumbers of locations with CoV-positive bats are depicted in bold. Distribution locations in the Netherlands are shown in Figure 1.
CoV, coronavirus.

GROUP 1, 2 CORONAVIRUSES IN URBAN BATS IN EUROPE
787

(Tylonycteris pachypus, China, HKU4); EF065509 (Pipistrellus
abramus, China, HKU5-1); EF065505 (P. abramus, China,
HKU5-5); DQ648819 (Pipistrellus pipistrellus, China, A434);
DQ648816 (Pipistrellus spp., China, 427); DQ648846 (Pipis-
trellus spp., China, A906); DQ648809 (P. abramus, China, 355);
DQ648811 (Pipistrellus spp., China, 363A).

Statistics

A number of bats (n) were sampled independently at ran-
dom (from the population of all bats at a location), such that in
the sampling method each bat had the same probability ( p) of
being a bat infected with CoV. The prevalence ( p) of CoV
in (sub)populations of bats over selected locations in the
Netherlands was estimated assuming this binomial process,
where p can be expressed as the total number of positive bats
(k) divided by n. The uncertainty about the estimated preva-
lence, due to sampling variability, is expressed using a beta
distribution (Vose 2000).

Results

Bat surveillance and CoV detection

Fecal samples were collected from a total of 265 bats and
analyzed for the presence of CoVs. Fifty-four bat samples
were omitted from the study due to the presence of RT-PCR
inhibiting factors. The remaining 211 samples represented 13
different bat species from 31 locations in the Netherlands
(Table 1 and Fig. 1). In total, 78.7% of the sampled bats were
adults. Females constituted 60.6% of the sampled bats, of
which 15.6% were juvenile and 14.8% were lactating adults.
Sixty-nine percent of the males were adults (Table 1).
The overall presence of CoVs in the bats was estimated at
16.9% (95% confidence interval ¼ 12.2–22.2). Four versperti-
lionid bat species were found positive for CoV, viz. M. dau-
bentonii, M. dascyneme, Nyctalus noctula, and P. pipistrellus,
with estimated prevalences of 17.3%, 19.6%, 37.5%, and

30.0%, respectively. No significant differences in the esti-
mated prevalences between the four bat species could be
observed (Table 2).
At 15 of the 31 locations (48.4%) bats were found positive
for CoVs. M. daubentonii was captured at 10 locations, of
which 5 yielded CoV-positive bats (50.0%). Respectively,
60.0% (6=10) and 50.0% (2=4) of the locations sampled for M.
dasycneme and P. pipistrellus were positive (Table 1). One lo-
cation (location 9) harbored both CoV-positive M. daubentonii
and M. dasycneme bats. No significant differences between
locations per species were observed (data not shown).
The presence of phenotypic properties predictive for CoV
infection in the Netherlands was investigated. None of the
analyzed factors (sex, age, and lactation=gravidity) were
predictive for CoV positivity—neither within the overall
sampling population (Table 3), nor within subpopulations
based on bat species (data not shown).

Group 1 and 2 CoVs in Dutch bats

The sequences of the PCR amplicons of 18 bat samples
could be determined and were analyzed phylogenetically.
Nucleotide sequence analysis showed that the bat-CoVs cor-
responding to the sequences amplified from Dutch bats are
members of group 1 and 2 CoVs (Fig. 2A, B, respectively).
Within group 1 five different lineages could be distinguished,
bringing the total number of group 1 lineages for European
bat-CoVs to 7 (Gloza-Rausch et al. 2008). Bat-CoV sequences
isolated from M. dasycneme formed two distinct clades within
group 1 (lineages 1 and 7). Each lineage was found at more
than one location, and at location 22 (Fig. 1) representatives of
both lineages were detected. The lineage 1 viruses clustered
together with bat-CoVs isolated from M. dasycneme in
Germany. Bat-CoVs associated with M. daubentonii clustered
together regardless of the sample locations and in a clade with
bat-CoVs isolated from the same bat species in Germany

Table 2. Estimated Prevalences of Coronavirus
in Four Positive Bat Species in the Netherlands

95% CI

CoV prevalence
Lower
Upper

Overall
16.9%
12.2%
22.2%
Species
M. dasycneme
19.6%
12.7%
27.6%
M. daubentonii
17.3%
8.4%
28.6%
N. noctula
37.5%
16.3%
61.6%
P. pipistrellus
30.0%
7.5%
60.0%

CI, confidence interval.

Table 3. Predictive Factors for Coronavirus
Detection in Bats in the Netherlands

Predictive
factor
Category
CoV
prevalence

95% CI

Lower
Upper

Sex
Female
15.4%
9.7%
22.0%
Male
20.0%
12.3%
29.1%
Age
Adult
14.9%
9.9%
20.6%
Female
15.4%
8.8%
23.4%
Female lactating
19.0%
5.7%
37.9%
Male
15.0%
7.2%
25.0%
Juvenile
25.5%
14.3%
38.8%
Female
18.2%
5.4%
36.3%
Male
33.3%
17.2%
51.8%

FIG. 2.
Phylogenetic analysis of CoV sequences found in bats in the Netherlands. Analysis based on a 334 bp fragment of
the RNA-dependent-RNA-polymerase as described before (Gloza-Rausch et al. 2008). Numbers at the nodes indicate boot-
strap values as percentage. Only values >70 are depicted. For all bat-CoVs putative reservoir species, country of origin, and
identification number are depicted. Common human CoVs are included for reference; Breda torovirus was used as outgroup.
CoVs identified in the present study are depicted in bold with the sampling location indicated between brackets. (A)
Phylogenetic analysis of group 1 Dutch bat-CoVs. The various phylogenetic groups and lineages within group 1 are indicated
on the right. (B) Phylogenetic analysis of group 2 Dutch bat-CoVs. The various phylogenetic groups and subgroups within
group 2 are indicated on the right.

‰

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789

(lineage 4). Bat-CoVs associated with N. noctula captured at
three different locations clustered together as well (lineage 5).
These data support the Chinese and German observations
that bat-CoVs are more closely associated with bat species
than with sample locations (Tang et al. 2006, Gloza-Rausch
et al. 2008). Both a group 1 (location 25) and a group 2 (loca-
tion 24) CoV was found to be associated with P. pipistrellus.
The group 1, lineage 6, CoV is most closely related to the
lineage 5 viruses associated with N. noctula, the group 2 CoV
clusters in putative subgroup 2c together with bat-CoVs
isolated in China (Fig. 2B). The nucleotide identities with the
Asian bat-CoVs in group 2c ranged from 64.96% to 97.57%.
The closest relationship is found with CoVs associated with
two Asian Pipistrellus species, viz. P. pipstrellus and P. abra-
mus. The nucleotide identities between Pipistrellus spp. asso-
ciated CoVs range from 79.22% to 97.57%.
M. dasycneme–associated Bat-CoV sequences within group
1 lineages 1 and 7 share nucleotide identities ranging from
97.61% to 99.71% and 97.63% to 99.63%, respectively, whereas
lineage 1 and 7 CoVs share nucleotide identities ranging from
71.07% to 72.59%. The lineage 7 M. dasycneme CoV sequences
are most closely related to a CoV isolated from a Rhinolophus
spp. sampled in the Shandong region in China (Tang et al.
2006), with identities ranging from 74.15% to 75.19%.
The bat-CoVs in lineages 1–4 and 7 are more related to
hCoV-229E and hCoV-NL63 than to the CoVs isolated from
N. noctula and the P. pipistrellus VM312 (nucleotide distance
matrix available on request).

Discussion

A grounded P. pipistrellus bat in the Netherlands in 2008
was found positive for a group 2 CoV. The identification of
P. pipistrellus as reservoir for group 2 CoVs is of importance
for the assessment of the zoonotic risks of these viruses. P.
pipistrellus is the most common and often most numerous
bat species in the Netherlands and Western Europe. It has a
preference for urban areas with roosting habits mainly in
buildings (Kapteyn 1997, Dietz et al. 2009), bringing it in
proximity to humans, thereby creating ample opportunities
for (in)direct bat–human contact. P. pipistrellus is mainly a
sedentary species and long-distance migration is rarely ob-
served. In the Netherlands P. pipistrellus is the bat species with
the most reported contact incidences with humans (Poel van
der et al. 2005). In fact this particular specimen was found by
humans in a residential quarter in a city.
The finding of group 2 CoVs in bats outside Asia supports
the opinion that group 1 and 2 bat-CoVs will circulate
worldwide, since recent evidence suggests that bat-CoVs are
the gene pools for all the mammalian CoVs in groups 1 and 2
(Vijaykrishna et al. 2007, Woo et al. 2009). Whether this par-
ticular virus circulating in the Netherlands can cause zoonotic
disease is not known. Further elucidation of its genome and
corresponding characteristics is needed.
Our study provided new evidence supporting the obser-
vation that bat-CoV lineages might be associated with bat
species rather than location. At location 9 both M. daubentonii
and M. dasycneme CoV-positive bats were present. Each bat
species was associated with a distinct CoV lineage that clus-
tered with CoVs isolated from the same species but at dif-
ferent locations, even at large geographic distances as, for
example, in Germany. No evidence for a common CoV cir-

culating between the two different bat species at location 9
was found. This reflects the local situation as both species
originate from different roost habitats and location 9 is just a
common foraging site for both species. This is indicative for a
strict species association rather than a limited local transmis-
sion. Bat-CoVs associated with N. noctula, here identified as
host for CoVs for the first time, captured at three different
locations clustered together as well.
P. pipistrellus and M. dasycneme bats appeared to host
phylogenetically distinct CoVs. The presence of both group 1
and 2 CoVs in one bat species has also been observed for R.
lechenaulti in China (Woo et al. 2006) and Hipposideros ruber in
Ghana (Pfefferle et al. 2009). In addition, M. ricketti and R.
ferrumequinum bats were found to be associated with more
than one CoV lineage (Tang et al. 2006, Woo et al. 2007). The
closest relationship of the group 2 CoV sequence isolated from
the Dutch P. pipistrellus was found with CoVs associated with
Asian bats from the same genus, viz. P. pipistrellus and P.
abramus (Tang et al. 2006, Woo et al. 2006). This close rela-
tionship of CoVs isolated from various species from the genus
Pipistrellus was not observed within group 1 CoVs. The group
1 CoV associated with a Dutch P. pipistrellus clustered in a
clade with the CoV sequences isolated from Dutch N. noctula
bats and not with the German CoVs associated with the two
other species in the genus P. natushii and P. pygmeaus. Mole-
cular evidence suggests that the genus Pipistrellus is not
monophyletic, and this might explain these differences (Jones
et al. 2002).
The estimated prevalence of CoVs in bats in the Nether-
lands was 16.9%. This is 1.7 times higher than the prevalence
determined in a German study sampling the same bat species
but with a limited geographic distribution (Gloza-Rausch
et al. 2008). Chinese studies sampling different bat species
yielded prevalences ranging from 4.2% to 16% (Chu et al.
2006, Tang et al. 2006, Woo et al. 2006, Lau et al. 2007). Forty-
eight percent of the sampled locations in the Netherlands
were positive. Positive locations were found in western,
eastern, and southern regions of the country, suggesting that
CoVs circulate widely in bat populations in the Netherlands.
To improve our knowledge about CoV circulation in bat
populations, we analyzed statistically the existence of pre-
dictive factors for CoV-positivity in bats. No predictive value
of age, sex, ongoing lactation=gravidity, species, or sampling
location for CoV detection was observed. In Germany, a
positive correlation was found with juvenile age and ongoing
lactation (Gloza-Rausch et al. 2008). A possible explanation
for the observed discrepancy could be the limited sample size
for some species and at some locations as reflected by the wide
confidence interval.
Our results clearly support other observations that CoVs
are diverse and widespread in bat species. Since there are
>1100 bat species worldwide that account for 20% of all
mammalian species, it is highly likely that the total extend of
the diversity of CoVs in bats has not been addressed yet.
Given the fact that (1) bats are considered as the natural hosts
for all mammalian CoVs, (2) (bat-)CoVs have a propensity for
interspecies jumping, and (3) certain bat species have habitats
in proximity to human dwellings, one should be aware of the
potential of bats as reservoirs for (highly) pathogenic zoonotic
CoVs. The identification of a group 2 CoV in a bat species
common to urban areas in most of Europe and notorious for
its close contact with humans underlines the need for vigi-

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REUSKEN ET AL.

lance. Therefore, the circulation of CoVs in bats should be
monitored closely (diversity and prevalence) and its zoonotic
potential should be investigated in more detail, including the
influences of bat diversity and behavior.

Acknowledgments

We are grateful to Hans Kramps, Wim van der Poel, and
Joke van der Giessen for helpful discussions. Anne-Jifke
Haarsma and Henriette van der Loo are acknowledged for
their assistance with sampling in the field. Katsuhishu
Takumi is thanked for his assistance with Figure 1.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Chantal B.E.M. Reusken
Laboratory for Zoonoses and Environmental Microbiology
Center for Infectious Disease Control
National Institute for Public Health and the Environment
P.O. Box 1
Bilthoven 3720BA
The Netherlands

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